Inappropriate T cell activation has been implicated in a number of deleterious conditions, including autoimmune diseases and transplant rejection. Optimal activation of T cells for clonal expansion is believed to require two signals. One is an antigen-specific signal delivered through T cell receptors (TCR), while the second is an antigen-non-specific or co-stimulatory signal.
The second non-specific signal is determined by a class of T cell regulatory molecules known as co-stimulators that determine whether T cells are activated or not. B7 (1 and 2), are co-stimulatory proteins expressed on the cell surface of antigen presenting cells such as macrophages, B lymphocytes and dendritic cells as reported.
B7 (1 and 2) is a natural ligand for T cell surface proteins known as CD28 and CTLA4 (cytolytic T-lymphocyte-associated antigen number 4, CD152). CD28 and CTLA4 share substantial homology in their amino acid sequences, particularly in the transmembrane and cytoplasmic domains, and are therefore believed to share similar functions in the T-cell co-stimulation pathway. B7 is known to have a greater affinity for CTLA4 compared with CD28.
CD28 is constitutively expressed on most T lymphocytes. CTLA4, however, was determined to be preferentially expressed by activated versus unactivated T cells.
The interactions of B7 with CD28 and CTLA4 play an important role in the full activation of T cells in the co-stimulation pathway during an immune response. Neutralisation of B7 or CD28 activity, for example with monoclonal antibodies, prevents T cell proliferation. Neutralisation of B7 activity also prevents T cells from acting as helper cells for the induction of antibody synthesis by B cells.
However, previous attempts to express the extracellular domain of CTLA4 as a soluble, unfused protein have been unsuccessful. According to PCT Publication No. WO 93/00431, successful expression of active CTLA4 proteins is believed to require an expression system that permits the proteins to form as dimers because the proteins are believed to occur in nature as dimers. Thus, researchers have focused on fusion proteins in an effort to find an appropriate expression system.
A fusion protein containing the extracellular domain of CTLA4 joined to the Fc heavy chain region of an immunoglobulin molecule has been developed as a possible agent having B7 inhibitory activity. This fusion protein, referred to as “CTLA4 Ig fusion protein,” is described in Linsley et al., J. Exp. Med. 174:561-569 (1991) and in PCT Patent Publication No. WO-A-93/00431.
According to these publications, the CTLA4-Ig fusion protein is secreted from mammalian cells as a disulfide-linked dimeric protein that aggregates in solution. However, attempts to express the extracellular domain of CTLA4 as an unfused protein in mammalian cells were unsuccessful. The Ig portion of the fusion protein facilitates the formation of a dimeric fusion protein, which is capable of being processed and expressed by the mammalian cells. The Ig portion additionally allows the active fusion protein to be purified from conditioned media using a protein A affinity column. Protein A has a high affinity for the Fc region of Ig molecules.
The molecular weight of the major CTLA4-Ig species in solution is approximately 200 kDa based on size exclusion chromatography also described in Linsley et al., J. Exp. Med. 174:561-569 (1991). Because the molecular weight of the monomeric form of the CTLA4-Ig fusion protein expressed in mammalian cells is about 50 kDa, the major species in solution is believed to be an aggregated complex of at least four CTLA4-Ig molecules.
The B7 inhibitory activity of the CTLA4-Ig fusion protein has been tested in both in vitro and in vivo experiments. In the in vitro experiments, the CTLA4-Ig fusion protein bound to B7 and neutralised its activity. The CTLA4-Ig fusion protein was found to inhibit T cell proliferation. The fusion protein also inhibited the ability of helper T cells to stimulate antibody production by B lymphocytes in an in vitro study described in Linsley et al., J. Exp. Med. 174:561-569 (1991).
In experiments conducted in vivo, the CTLA4-Ig fusion protein was determined to be immunosuppressive and capable of prolonging survival of pancreatic and heart allografts in mice and rats. In mouse studies, the administration of CTLA4-Ig resulted in the long term acceptance of allografts. These results suggest that the fusion protein had tolerized the recipient mice to the foreign tissue.
The CTLA4-Ig fusion protein has several disadvantages as a therapeutic agent for human disease. Because the fusion protein is a non-naturally occurring molecule, a patient receiving the protein may develop an immune response to the protein.
Antigenicity may be more of a problem when patients are taken off the therapeutic agent so they are no longer immunosuppressed and are capable of mounting an immune response against the fusion protein. Therefore, antigenicity may prevent the CTLA-Ig fusion protein from being administered intermittently to patients suffering from chronic diseases. In addition, the half-life of the CTLA4-Ig fusion protein in mice is about 4 days, with significant levels of the fusion protein still detectable in the animals 5 weeks after the cessation of treatment with CTLA4-Ig. Furthermore, when bound to B7 on the surface of antigen-presenting cells, the Ig portion of the fusion protein may activate the complement cascade that results In cell death and hematological problems.
Thus, a need exists for additional therapeutic agents capable of inhibiting the co-stimulatory pathway in T cell activation. The present invention satisfies this need and provides related advantages as well.